我们是从美国国立卫生研究院（奖励R00 NS072192 MKL，HD029178到AS.L.，2 RO1 NS032457到CAW）的支持表示感谢。 MKL是波士顿​​儿童医院的职业发展奖学金/哈佛医学院岸奖学金和研究员Alfred P. Sloan基金会的收件人。 CAW是霍华德·休斯医学研究所的研究员。

1。胚胎分离/准备工作这种技术可以用于小鼠或大鼠。在这个协议中，我们展示了CSF采集技术，大脑皮质的外植体解剖小鼠胚胎脑。我们会就任何重要的差异大鼠与小鼠存在于一般技术。为胚胎的年龄分期系统，E1被归类为天大鼠插头，E0.5被归类为对小鼠的插头的天。 <ol><li>准备一个用Sylgard有机硅弹性体的显微解剖盘。 Sylgard的184有机硅弹性体提供液体成分由两部分组成，第一部分A和B部分，A部分和B部分混合在按重量或体积的比例为10:1。液体组分混合后，倒入一个Petri培养皿中倒入Sylgard的弹性体，以覆盖整个表面的菜。有些气泡可能是本，将在固化过程中消散。将陪替氏培养皿上的盖子，并允许elastom呃治愈。该弹性体可以在室温下固化24小时，或在较高的温度（ 例如 37℃）为更快的固化。一旦液体成分凝固，解剖盘就可以使用了。可反复使用的菜多个实验，只要它被清洁以及下面的过程。 </li><li>的微毛细吸管（针）的制备。制备微毛细吸液管通过施加热和拉使用一个成茂PC-10垂直的微量吸移管拉出器具有以下设置:一个步骤拉加热器＃2设置为58;100克拉砝码。精尖的微管是用细＃55镊子小心折断。针将得到的平均内径为85微米。 </li><li>准备抽吸器组件为CSF愿望。插入微柱塞设置有微毛细吸液管进入毛细管针。或者，将一个塑料的一次性过滤器的端部的吸气管组件，连接吨Ø微毛细吸管。抽吸器组件的另一端的位置上的垫片插入推针贯通。 </li><li>把胚胎从垃圾中分离到微解剖盘准备用Sylgard。 </li><li>移除额外的胚胎外膜和组织，以便清楚地暴露的胚胎。每个组织层第一的子宫壁，然后蜕膜可以解剖用细的虹膜切除剪刀（ 即精细的科学工具＃15000-02）。每个植入部位，首先子宫壁，然后蜕膜可以切开子宫的长轴平行的，并然后被打开切口可进一步用细镊子。蜕膜后，可以删除一个类似的切口，露出胎膜。使不切开或刺破胎膜，应谨慎行使。 </li><li>用无菌Hanks平衡盐溶液（HBSS）清洗，并从周围的胚胎，使用移液管，以及一个kimwipe或过滤器除去过量的流体纸剪成三角形。 </li></ol> 2。心室CSF收集<ol><li>可视化解剖显微镜下鼠标，胚胎应在其一侧铺设，有了一个矢状发育中的胚胎的胚胎。用大鼠胚胎E16或以上的位置，在其背部脊柱的胚胎，沿其最长的平面尺寸，从颅尾方向，在它的后面，如果胚胎是在撒谎。以这种方式收集CSF可以从右侧和左侧侧脑室。 </li><li>稳步微毛细吸管插入到侧脑室，脑脑室或小脑延髓池，试图不接触神经上皮细胞一旦已插入吸管。对于小鼠胚胎E14.5或更旧，被吸出的CSF可以自右心室，然后除去针插入左心室。由于脑室和胚胎的神经管的通畅年龄小于E14.5，整个CSF的体积往往可被吸出从与一个单一的针插入侧脑室。然而，这并非总是如此发育的时间和连接脑室通畅可能略有不同，因此，微毛细吸管也可以被插入到小脑延髓池收集最大CSF体积。 </li><li>一旦微毛细吸管插入侧脑室，中脑脑室或小脑延髓池，仔细，轻轻地开始CSF进入移液管吸移，使用微柱塞创建负压抽吸的CSF，或通过提供一种温柔负压口使得CSF开始轻轻流入微毛细管的吸移管在一个缓慢的和受控的方式创建的。我们建议，观众会与有关机构的政策，这些方法自己的地方官员。 </li><li>继续施加负压，并收集到微毛细吸管的CSF。在小鼠和大鼠胚胎为E16.5/E17或更年轻的，它是可能的轻微上的头部侧的微毛细吸管英寸在较旧的胚胎形成的草皮层的外观，观察心室崩溃，由于大脑的较大尺寸的，它可能不会是可能观察头部的崩溃。 </li><li>停止施加压力，并温和地去除微毛细吸管从侧脑室。 </li><li>轻轻驱逐CSF样品放入Eppendorf管中，已在冰上冷却。 </li><li>从整个一窝动物继续收集CSF和游泳池的样品为同一管。 </li><li>在4℃下以10,000 xg离心10分钟，以除去任何污染的细胞。 </li><li>分析样品的污染神经上皮细胞或红血细胞的任何迹象。污染的迹象，通常表示由流体出现混浊或红/粉红色，或颗粒的存在下，用带血点。离心分离后，将CSF可以通过显微分析的细胞或细胞碎片的任何证据。如果有迹象污染的流体应该被丢弃。作为一个额外的控制，也可被染色的颗粒的含量为细胞。一个干净的CSF样品可用于细胞培养，皮质文化，法，免疫印迹，ELISA，和其他实验。明确CSF应被转移到一个新的无菌Eppendorf管中。现在可以用于分析样品可以与其他样品用液氮冷冻，或管理单元，汇集，并储存在-80℃下冻结和解冻的流体的小样本，可能会导致在小体积减少和蛋白浓度的变化。这可避免通过冷冻干燥的样品。 </li></ol> 3。皮质外植体解剖<ol><li>传输的E14.5胚胎分离出的垃圾到微解剖菜准备用Sylgard。 </li><li>移除胚胎从胚外膜和组织，如在步骤1.5所述。 </li> &lt;李&gt;洗无菌Hanks平衡盐溶液（HBSS），并除去过量的流体，用移液管从周围的胚胎。 </li><li>解剖从胚胎的其余部分（ 图1A）中分离的头部穿过宫颈区域。 </li><li>使用精细的手术刀眼科刀，切下来的中线的头皮。抓住每边用细镊子此切口，去除皮肤。接下来，使用虹膜切除术剪刀做一个切口，运行中线的发展头盖骨的长度。随后，使两个额外的切口，约1/3的距离，从显影颅骨的前部和后部的端部。用细镊子把握"襟翼"的头盖骨，这部分的解剖结果，并拆下显影的头盖骨组织，暴露皮层（ 图1B）。 </li><li>使用手术刀沿中线平分线大脑中期矢状平面中，分离的右侧和左侧的皮质半球（ 图1B，C）。软脑膜和蛛网膜装的皮质，硬脑膜与发展头骨一起被除去。 </li><li>单独准备每个皮质半球。将半球的内侧，以便您可以看到神经节隆起和发展皮层（ 图1C，D）。 </li><li>使用手术刀，使冠状切口，通过大脑皮质神经上皮尾侧嗅球的发展。这的切口应该开始前边界的外侧和内侧神经节隆起，穿过前扣带回区域发展的皮质雏形（ 图1E）。 </li><li>另一个的冠状切口尾部的后边界的横向神经节隆起（ 图1F）。此切口也应该从神经节隆起的侧边界的内侧壁皮质雏形。 </li><li>收回皮质的"啪啪"CREA通过在步骤3.7和3.8的两个切口，使用手术刀或温和的压力从一个流中，用200微升移液器交付的HBSS中，以避免机械损伤的皮质。然后作一横切口，沿边界分离的的横向神经节隆起，从发展皮质（ 图1G，H）。然后，剖析了发展中的海马和大脑皮质的下摆内侧前脑，在新皮层外侧皮质表面符合半球间墙的顶点。 </li><li>作一横切口，沿边界分离的的横向神经节隆起，从皮层（ 图1H）。 </li><li>删除任何额外的Sylgard的板从解剖皮质外植体解剖组织。可以使用温和的移液管移液与新鲜的HBSS缓冲的任何额外的解剖组织。解剖的的皮质是现在有脑膜面朝下（ 图1I）。 </li></ol><ol><li>准备的铂丝的玻璃移液管连接到环路。加热的玻璃移液管与插入的玻璃移液管的端部中的折叠的铂丝。通过加捻的铂丝圈的端部，在循环的大小可以增加或减少，循环可以被成形为适应所需的外植体的大小。 （这可以预先准备和重复使用。） </li><li>的铂丝的端部加热到消毒循环。 </li><li>隔离解剖的皮质外植体并删除额外的的HBSS媒体的移液温和，留少量使用的钢丝环。 </li><li>将1厘米聚碳酸酯膜在4厘米直径的成像碟。 </li><li>使用侧面的铂丝圈，轻轻翻转，使皮层脑膜的一面朝上。 </li><li>关闭夹层皿中，将外植体在循环中间放置和使用的固有的静水压力抬起皮层的外植体电梯外植体在平面上。 </li><li>轻轻地放置在所述聚碳酸酯膜的外植体，并解除铂丝圈离开的外植体，使得仍然心室与膜的表面接触的平坦的平面有上的膜。 </li><li>轻轻抬起膜和膜下吸取50μL的CSF或其它媒体。 </li><li>量的摄像培养皿，将其放置在湿润的副容器，并在5％CO 2在37℃下孵育24小时，以支持持续的细胞增殖和外植体生长。 图2示出了示意性的最终的皮层外植体的培养皿制备。 </li></ol>

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作者宣称，他们没有竞争的金融利益。

心室CSF收集所描述的方法，已经取得了相当纯的胚胎脑脊液样品与在一些细胞分析9的稳定的蛋白质组合物，和一致的活性。有了一个良好的采集技术和产仔数的10 E14.5小鼠，可以预期的CSF收集10-15微升，并从E16鼠一窝，谁也无法预料的CSF收集约50-90微升。此收集技术，从血液和组织中的污染最小化，通过仔细观察，离心分离，和废弃的样品与细胞碎片或血色调的任何证据。我们已经分析了?...

CSF是一种复杂的流体，沐浴神经上皮1-6，并提供必要的压力7和促进生长发育中的大脑8-12线索。研究CSF在大脑发育过程中，我们开发的技术隔离室CSF来自发展中国家的大鼠或小鼠胚胎过程中各阶段的发展6,9。往前的隔离的方法包括用玻璃微针和隔离的CSF使用微喷射器1,2。我们的方法利用玻璃微毛细吸管的尖端一直拉到创建一个超细点，以提高组织的渗透。玻璃微毛细吸管被连接到一个吸气器与温和的压力变化，使心室CSF可以控制收集。为了研究干细胞的的CSF信号的影响，我们解剖大脑皮质外植体，将它们放置在聚碳酸酯膜，并把它们飘浮在适当的铜CSF样品9 lture培养基中。利用这种技术，减少体积的介质来培养的组织，是足够允许脑脊液9的有效利用。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Wiretrop II毛细管针</td><td>德拉蒙德科学</td><td> ＃5-000-2020 </td><td></td></tr><tr><td> SYLGARD </td><td>安士澳粘合剂</td><td> ＃184 SIL ELAST包的</td><td></td></tr><tr><td>吸气管组件</td><td>西格玛</td><td> ＃A5177-5EA </td><td></td></tr><tr><td>一次性过滤器</td><td>文丘里或当地的药店</td><td>不可用</td><td>标准香烟过滤嘴</td></tr><tr><td> Roundstock眼科刀（15度捅一刀） </td><td>世界精密仪器公司</td><td> ＃500250 </td><td></td></tr><tr><td> 35毫米玻璃底培养皿</td><td> MatTek公司</td><td> ＃P35G-1.5-14-C </td><td&gt; </td></tr><tr><td>铂铱丝</td><td> Tritech公司研究</td><td> ＃PT-9010-010-3FT </td><td></td></tr><tr><td>核孔径迹蚀刻膜</td><td> Whatman公司</td><td> ＃09-300-57 </td><td></td></tr><tr><td> Hanks平衡盐溶液</td><td> Fisher Scientific则</td><td> ＃SH30031.FS </td><td></td></tr><tr><td>虹膜切除术剪刀</td><td>精细科学工具</td><td> ＃15000-02 </td><td></td></tr></tbody></table>

isolation of cerebrospinal fluid from rodent embryos for use with dissected cerebral cortical explants

CSF是一种复杂的流体动态变化的蛋白质组的整个开发过程，并在成年后。在胚胎发育过程中，新生的CSF区别于前神经管闭合时的羊水。 CSF卷，然后在随后的日子里，排队的神经上皮干细胞的心室和脉络膜丛产生CSF增加。胚胎CSF接触的顶端，心室发育中的大脑和脊髓神经干细胞的表面。 CSF提供了重要的流体压力发育中的大脑的扩展和分发重要的增长推动因素，神经祖细胞在时间上的具体方式。要调查的CSF的功能，重要的是不从血液或显影端脑组织污染隔离胚胎脑脊液的纯样品。在这里，我们描述了一种技术，以隔离的心室胚胎CSF可用于相对纯的样品质谱，蛋白质电泳，和细胞与初级外植体培养的实验分析，包括范围广泛的。我们演示了如何剖析和文化多孔聚碳酸酯膜的皮层外植体对以成长发展的皮层组织与媒体或CSF的量的减少。使用这种方法，可以进行实验，使用CSF从不同年龄或条件调查对靶细胞的CSF蛋白质组的生物活性。

CSF集合应得到一个明确的，透明的液体。不应该有任何从血液中的污染的证据证明，抽吸，并在Eppendorf管中的流体由一个红色或黄色带点。此外，也应该没有证据表明组织中吸出物和Eppendorf管。当CSF离心，也可以评估的CSF显微镜，以确保有没有污染。如果有污染的迹象时，CSF应该被丢弃。从E14.5鼠标乱抛垃圾，一个可以预见收集10-15微升CSF。 表1描述了从不同胚龄小鼠和大鼠的平均窝?...

Watch this Scientific Journal Video about Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants at JoVE.com

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

心室脑脊液（CSF）沐浴在早期的大脑发育在胚胎神经上皮和大脑皮质祖细胞。在这里，我们描述的方法，从不同年龄层的啮齿动物胚胎隔离室CSF，为了研究其生物学功能。此外，我们展示了我们的脑皮质外植体的解剖和文化的技术，可以为外植体生长的培养基或CSF以最小的体积。

脑脊液分离解剖大脑皮质外植体使用的啮齿动物胚胎

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

isolation-cerebrospinal-fluid-from-rodent-embryos-for-use-with

Research

JoVE Journal

Neuroscience

22.9K 次观看.  VA Greater Los Angeles Healthcare System. 心室脑脊液（CSF）沐浴在早期的大脑发育在胚胎神经上皮和大脑皮质祖细胞。在这里，我们描述的方法，从不同年龄层的啮齿动物胚胎隔离室CSF，为了研究其生物学功能。此外，我们展示了我们的脑皮质外植体的解剖和文化的技术，可以为外植体生长的培养基或CSF以最小的体积。

视频: Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

科研

神经科学

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

前子宫穿孔和整个半球的外植体早期皮质发育的研究:一个简单的实验方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

分离及脑实质小动脉插管

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

人脑脊液 Endopeptidomic 分析的样品制备

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

轻度宫内灌注圈狭窄的大鼠模型

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

一种改进的麻醉小鼠脑脊液采集方法

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

由亲炎细胞因子的内脑注射诱导的广度脑皮质脱叶的鼠模型

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...